Severity markers in severe leptospirosis: a cohort study.

We aimed to evaluate parameters for their value as severity markers in hospitalized leptospirosis patients. We recruited 47 informed adult consenting patients and assessed a number of clinical, hematological, biochemical, and biological variables. Patients were sorted according to severity based on fatality or the requirement of mechanical ventilation or dialysis; the parameters studied were compared between groups on inclusion and the next day. Beside septic shock presentation or a high severity score (Simplified Acute Physiology Score; SAPS II), increased lactate, total bilirubin, lipase, and AST/ALT ratio or a decreased cytokines IL-10/TNF-α ratio were all significantly associated with severity. The gene expression of the IL-1 receptor antagonist IL-1ra, IL-1α, and the long pentraxin PTX-3 were also transcribed at higher levels in most severe cases. Patients could rapidly improve or deteriorate, highlighting the need for a new assessment the next day. Our results add to the limited body of knowledge about severity markers in leptospirosis. They also suggest that patients should be reassessed the next day before being possibly discharged from the hospital. Further studies are needed in order to confirm relevant and reliable prognostic parameters in leptospirosis that would be helpful for the purpose of triage.

Pharmacokinetics of the angiotensin-converting-enzyme inhibitor, benazepril, and its active metabolite, benazeprilat, in dog.

1. The pharmacokinetics of the angiotensin-converting-enzyme (ACE) inhibitor benazepril were evaluated in eight healthy Beagle dogs. Benazepril was administered orally at a dosage of 7.5 mg (about 0.5 mg/kg) both as a single dose and then once daily for 14 consecutive days. The prodrug, benazepril, and its active metabolite, benazeprilat, were measured in plasma using a gas chromatography mass-spectrometry method with mass-selective detection. 2. Benazepril appeared quickly in the plasma (tmax 0.5 h) and was rapidly eliminated by metabolism to benazeprilat. Peak benazeprilat concentrations were attained later (tmax 1.25 h) and declined biphasically with a rapid elimination phase (t1/2 lambda 1 1.1 and 1.7 h after single and the last repeated dose respectively) followed by a terminal elimination phase (t1/2 lambda z 11.7 and 19.0 h after single and repeated dose respectively). The mean residence time for benazeprilat was 15.2 h after the single dose and 17.4 h after the 14th dose. 3. Repeated administration of benazepril produced moderate bioaccumulation of benazeprilat; the ratio of AUC[0-->24 h]'s after the 14th dose as compared with the single dose was 1.47, equivalent to a half-life for accumulation (t1/2acc) of 14.6 h. Steady-state benazeprilat concentrations at peak (Cmax) and trough (Cmin) were reached within three doses. 4. The pharmacodynamics of benazepril were assessed by measurement of plasma ACE activity. After both single doses and at steady-state, benazepril produced inhibition of ACE activity in all dogs that was maximal at peak effect (Emax = 100%) and long-lasting (> 85% inhibition was present at 24 h). The long duration of action of benazepril on plasma ACE is due to the presence of the terminal elimination phase of benazeprilat, even though most of the metabolite is rapidly eliminated from the plasma.

Pharmacokinetics of the active metabolite of benazepril, benazeprilat, and inhibition of plasma angiotensin-converting enzyme activity after single and repeated administrations to dogs.

Plasma pharmacokinetic variables of benazeprilat, the active metabolite of the angiotensin-converting enzyme (ACE) inhibitor benazepril, were evaluated in healthy Beagles. Benazeprilat was administered IV at a dosage of 0.5 mg/kg of body weight (n = 9). The elimination of half-life of benazeprilat was 3.5 hours, although an additional terminal phase was observed in some dogs. Vehicle (gelatin capsules) or benazepril at dosages of 0.125, 0.25, 0.5, or 1.0 mg/kg was administered orally as a single administration, then once daily for 15 consecutive days (n = 5 or 6/group). Peak benazeprilat concentrations were rapidly attained by 2 hours. Benazeprilat concentrations accumulated moderately with repeated administration, with a peak concentration that was 23% higher and an area under the concentration-time curve that was 34% higher after the 15th dose of benazepril, compared with values after a single dose. The effective half-life for accumulation for all 4 dosages was 12 hours. Steady-state concentrations at 2 hours after administration were achieved after a median (range) of 1 (1 to 6) dose(s). Pharmacodynamic variables were assessed by measurements of plasma ACE activity after oral administration of benazepril or vehicle. All dosages of benazepril caused profound inhibition of ACE, with rapid onset of activity (time to peak effect, 2 hours) and long duration of action (single administration of all 4 doses induced inhibition of ACE that was significantly different from the value in the control [vehicle-treated] dogs for all time points between 1 and 30 hours). Maximal inhibition at all time points was induced by the 0.25-mg/kg dosage at a single administration and with the lowest dosage tested (0.125 mg/kg) at steady state. At steady state, the 0.25-mg/kg dosage caused (mean +/- SEM) 96.9 +/- 2.0% inhibition of ACE activity at maximal effect and 83.6 +/- 4.2% at trough effect (24 hours after dosing), indicating minimal variation in peak/trough effect. Steady-state inhibition of ACE activity at both peak and trough drug effect was achieved after 1 to 4 doses. The data indicate that benazepril is a potent and long-acting ACE inhibitor in dogs.

High performance liquid chromatography and pharmacokinetics of the non-steroidal anti-inflammatory drug oxindanac in calves.

A high-performance liquid chromatographic method for the determination of the non-steroidal anti-inflammatory drug, oxindanac, in calf plasma is described. Recoveries over the concentration range 0.375 to 62.5 micrograms/ml were 90.2-107.8% with interassay coefficients of variation of 2.1-22.3%. The limit of detection was estimated as 0.10 micrograms/ml and the limit of quantification calculated to be 0.24 micrograms/ml in a 1 ml plasma sample. This method was used to establish the pharmacokinetics following intravenous (i.v.), intramuscular (i.m.) and oral (p.o.) administration to calves of oxindanac at a dose rate of 2 mg/kg. The elimination t1/2 was long (t1/2 21.2 h after i.v. injection) and absorption was rapid (t1/2a 0.072 h) and complete (F > 100%) following i.m. administration. Bioavailability was incomplete (F = 66.6%) following p.o. administration to calves that had been fed on milk, and Wagner-Nelson analysis revealed two absorption phases (t1/2's 0.20 and 1.9 h). Oxindanac produced long-lasting inhibition of serum TxB2 production, with mean Emax values (% inhibition) of 96.8, 94.1 and 81.3 following i.v., i.m. and p.o. administration, respectively. A single i.v. or i.m. injection of 2 mg/kg oxindanac will probably be active in calves for at least 36-48 h.

Bidirectional chiral inversion of the enantiomers of the nonsteroidal antiinflammatory drug oxindanac in dogs.

The nonsteroidal antiinflammatory drug oxindanac exists as two enantiomers, with most of its pharmacological activity residing in the (S)-isomer. The behavior of its enantiomers was investigated in dogs. Bidirectional inversion occurred in heparinised plasma and blood, with a ratio of enantiomers [S:R] of 7.3:1 being achieved at equilibrium after incubation for 24 h at 37 degrees C. There was no detectable inversion of either isomer in plasma incubated at 4 degrees C for up to 8 h or in aqueous solution at 37 degrees C for up to 36 h. Bidirectional inversion also occurred in vivo, with a ratio of plasma AUC (0 infinity)s [S:R] of 8.1:1. The ratio of enantiomers reached equilibrium within 2 hr following (S)- or rac-oxindanac, and within 8 h following (R)-oxindanac. Elimination t1/2s of the isomers were the same (R, 12.1 h, S, 13.3 h). There were no differences in the ratio of enantiomers following oral or intravenous application, suggesting that a systemic site for inversion was predominant. Although concentrations of the respective isomers were similar at equilibrium following administration of either (R)-, (S)-, or rac-oxindanac, AUC (0 infinity)s differed due to the delay in reaching equilibrium. The extent of inversion to the (S)-isomer was 100, 73.2, and 60.7% after administration of (S)-, rac-, and (R)-oxindanac, respectively. Although pharmacological activity might be equivalent at equilibrium following administration of either (R)-, (S)-, or rac-oxindanac; efficacy at early time points should be superior in the order (S) > racemate > (R).(ABSTRACT TRUNCATED AT 250 WORDS)

Choline increases acetylcholine release and protects against the stimulation-induced decrease in phosphatide levels within membranes of rat corpus striatum.

This study examined the possibility that membrane phospholipids might be a source of choline used for acetylcholine (ACh) synthesis. Slices of rat striatum or cerebellum were superfused with a choline-free or choline-containing (10, 20 or 40 microM) physiological solution with eserine, for alternating 20 min periods of rest or electrical stimulation. Superfusion media were assayed for choline and ACh, and slice samples taken before and after stimulation were assayed for choline, ACh, various phospholipids, protein and DNA. The striatal slices were able to sustain the stimulation-induced release of ACh, releasing a total of about 3 times their initial ACh contents during the 8 periods of stimulation and rest. During these 8 cycles, 885 pmol/micrograms DNA free choline was released from the slices into the medium, an amount about 45-fold higher than the initial or final free choline levels in the slices. Although repeated stimulation of the striatal slices failed to affect tissue levels of free choline or of ACh, this treatment did cause significant, dose-related (i.e., number of stimulation periods) stoichiometric decreases in tissue levels of phosphatidylcholine (PC) and of the other major phospholipids; tissue protein levels also declined significantly. Addition of exogenous choline to the superfusion medium produced dose-related increases in resting and evoked ACh release. The choline also fully protected the striatal slices from phospholipid depletion for as many as 6 stimulation periods. Cerebellar slices liberated large amounts of free choline into the medium but did not release measurable quantities of ACh; their phospholipid and protein levels did not decline with electrical stimulation. These data show that membrane phospholipids constitute a reservoir of free choline that can be used for ACh synthesis. When free choline is in short supply, ACh synthesis and release are sustained at the expense of this reservoir. The consequent reduction in membrane PC apparently is associated with a depletion of cellular membrane. The use of free choline by cholinergic neurons for two purposes, the syntheses of both ACh and membrane phospholipids, may thus impart vulnerability to them in situations where the supply of free choline is less than that needed for acetylation.

Phosphatidylcholine as a precursor of choline for acetylcholine synthesis.

It has been hypothesized that the selective vulnerability of certain brain cholinergic neurons in Alzheimer's disease may reflect the unique way that choline is utilized by these neurons, i.e. not only as a component of major membrane phospholipids, e.g. phosphatidylcholine (PC), but also as a precursor of their neurotransmitter, acetylcholine (ACh). A prolonged utilization of choline liberated from PC, for ACh production, without adequate resynthesis of this lipid, might result in a net loss of the phosphatide followed by an impairment of membrane function and loss of cellular viability. Studies described in this paper, performed on electrically stimulated striatal slices and on cholinergic cell lines, test this hypothesis. 1) Electrically-stimulated striatal slices continue to release ACh, and sustain their free choline and ACh levels, even when perfused with a choline-free medium. Striatal levels of PC decline under these circumstances, and this decline can be blocked by adding tetrodotoxin (which blocks neuronal depolarization) or choline to the medium. The other major membrane phospholipids, phosphatidylserine and phosphatidylethanolamine, also decline proportionately to PC when slices are stimulated in the absence of choline. 2) In a population of purely cholinergic cells (human neuroblastoma, LA-N-2), ACh can be synthesized from choline derived from degradation of endogenous PC formed de novo by methylation of phosphatidylethanolamine. 3) PC content of cells in culture (neuroblastoma X glioma hybrid, NG 108-15) can be altered by adding various amounts of choline to the growth media. The proportion of PC in the cells apparently affects cellular survival and rate of growth. Taken together these data demonstrate that cholinergic neurons utilize the choline stored in PC to synthesize ACh; that this process may lead to a depletion in membrane phospholipids (when choline supply is inadequate); and that the resulting changes in neuronal membrane composition might adversely affect cellular viability.

Evidence for the presence of a neutral insulinotrophic peptide in the porcine duodenum.

A crude mixture of thermostable peptides extracted from porcine duodenum was fractionated by electrofocusing. A neutral fraction, different from the basic fractions of GIP, VIP, PHI, and CCK was found to promote insulin secretion when injected in vivo to normal rats. This neutral fraction, extracted from the crude mixture by chromatography, stimulated insulin output from an isolated rat pancreas and enhanced glucose-induced insulin release. The insulinotrophic effect of this partially purified duodeno-jejunal material disappeared following digestion with trypsin. The insulin-releasing activity was found to correspond to a compound of molecular weight higher than that of insulin (i.e. higher than 6000). No GIP-like immunoreactivity was found in this neutral fraction indicating that the active peptide(s) are not GIP related compounds. These observations suggest that porcine duodenum contains and incretin activity different from that of the insulinotrophic factors already reported.

The treatment of obesity by carbohydrate deprivation suppresses plasma tryptophan and its ratio to other large neutral amino acids.

We measured plasma concentrations of tryptophan (Trp) and the other large neutral amino acids (LNAA) in 6 control and 7 obese subjects before and after they consumed a low-carbohydrate "protein-sparing modified fast" (PSMF) diet; LNAA levels in control subjects were also assessed after supplemental oral Trp. Consumption of the PSMF diet by non-obese subjects, or obesity per se, caused major reductions in the ratio of the plasma Trp concentration to the summed plasma concentrations of the other LNAA (i.e., the "plasma Trp ratio"), and may thus have diminished brain serotonin synthesis. Administration of even 2 g of supplemental Trp did not elevate the plasma Trp ratio beyond the normal range observed previously in subjects consuming carbohydrate-containing meals.

Effect of various oral glucose doses on plasma neutral amino acid levels.

Six healthy, nonobese, fasting subjects each received, on different days 0, 6, 12.5, 25, or 50 g of glucose (Glucola) in a total volume of 100 ml. Blood was taken at intervals and assayed for plasma levels of the branched-chain amino acids (valine, isoleucine and leucine); the other major large neutral amino acids (LNAA) (methionine, phenylalanine, tyrosine and tryptophan); and, in some cases, insulin and glucose. Insulin levels were significantly elevated 30 min after consumption of 12.5, 25, or 50 g of glucose, and were higher after the 50 g dose than after 12.5 g. Changes in plasma glucose concentrations were small and did not correlate with glucose dose. Mean percent reductions of LNAA tended to exhibit dose-dependence, most clearly observed after 120 min. In some subjects as little as 6 g of glucose transiently decreased LNAA concentrations. Branched-chain amino acids were most sensitive, decreasing by 35%-41% after 50 g of glucose. Plasma tryptophan concentrations fell only by 23%, hence the ratio of plasma tryptophan to other plasma LNAA (which affects brain serotonin synthesis) increased significantly.

Developmental changes in brain indoles, serum tryptophan and other serum neutral amino acids in the rat.

The rates at which brain neurons synthesize and release serotonin depend in part on brain tryptophan concentrations; these, in turn, vary directly with serum (or plasma) tryptophan, and inversely with the serum concentrations of other large neutral amino acids (LNAA). Concentrations of serum tryptophan, LNAA and brain indoles were examined in samples drawn at noontime from rats aged 0-59 days. Developmental changes in serum tryptophan largely paralleled those in the tryptophan/LNAA ratio, and brain tryptophan concentrations. Brain serotonin and 5-hydroxyindole acetic acid (5-HIAA) levels also increased postnatally; the changes in 5-HIAA tended to parallel those in brain tryptophan while those in serotonin did not.

Morphine analgesia in grouped and isolated rats.

The effects of long-term isolation of young adult male rats on the analgesic effects of morphine were investigated. Isolated rats developed altered patterns of behavior, including muricidal behavior in some animals. Analgesic activity of morphine was assessed with both the tail compression and the hot plate methods. The results indicate that chronically isolated rats, whether developing muricidal behavior or not, show no alteration in either pain thresholds or in their response to morphine-induced analgesia.